Recent advances in the ability to construct arrays of biological compounds have greatly facilitated the ease and speed with which certain biological assays can be performed. For example, in the areas of nucleic acid sequencing and analysis, the advent of new technologies for constructing arrays of immobilized target nucleic acids or oligonucleotide probes has enabled the rapid screening and sequencing of nucleic acids. Arrays of peptides and small biomolecules have also proven useful in binding assays used in pharmaceutical development. The usefulness of these arrays depends on the ability to generate and use arrays with spatially addressable regions of defined composition or sequence.
Several technologies have been developed for producing such arrays. For example, several researchers have devised methods for in situ synthesis of arrays of biological polymers, such as nucleic acids, peptides, and carbohydrates. These methods use, for example, physical barriers to separate different synthesis sites, devices (such as inkjet printers) for precise delivery of reagents to different synthesis sites, or masking techniques that allow the use of light to determine the course of synthesis. See, e.g., WO 90/03382; Fodor et al., 1991, Science 251:767-73; Pease et al., 1994, Proc. Natl. Acad. Sci. 91:5022-26; U.S. Pat. No. 5,424,186, to Fodor et al. Alternatively, presynthesized biological compounds or biological polymers may be attached directly to the substrate at precise positions using a variety of techniques, ranging from simple spotting to robotic delivery systems. A variety of different substrates and techniques for attaching the biological compounds to the substrates are also available.
As noted above, arrays of nucleic acids have proven particularly valuable. The ability to perform many previously available techniques has been greatly enhanced by the availability of arrays, which permit many assays to be performed simultaneously, rather than having to do each assay individually. Other techniques that would have been virtually impossible are now possible using polynucleotide arrays.
One technique that has been particularly enhanced by the availability of arrays of nucleic acids is sequencing by hybridization (SBH). SBH is a technique for rapidly sequencing nucleic acids without using gels. In SBH, polynucleotides having overlapping sequences are hybridized to a target nucleic acid. The sequences of the polynucleotides that hybridized are determined and their common sequences overlapped to generate the sequence of the target nucleic acid. The use of arrays has allowed the generation of sufficient hybridization information to make SBH feasible on a large scale.
SBH is divided into three formats, depending on the nature of the array and the way in which it is interrogated. In Format I, an immobilized target nucleic acid is interrogated with labeled solution-phase polynucleotide probes. In Format II, a spatially-addressable array of immobilized polynucleotide probes is interrogated with a labeled solution-phase target nucleic acid. In Format III, an array of immobilized polynucleotide probes is hybridized with an unlabeled solution-phase target nucleic acid and one or more labeled solution-phase oligonucleotide probes. Hybridization is assayed by ligating the labeled oligonucleotide probes to the immobilized polynucleotides. All three formats require the ability to distinguish perfectly matched hybrids from hybrids that contain a single mismatch at any position. For a more detailed discussion of SBH and the three formats, see WO 98/31836, particularly at pages 1-3.
While the availability of high-density arrays of immobilized compounds has revolutionized the speed with which certain biological assays can be performed, array-based assays still suffer from drawbacks. Samples are often available in limited amounts, which are incompatible with the large volumes of assay solutions required to immerse the arrays. Thus, there remains a need in the art for improved arrays that allow the use of small volumes of assay solutions.